ES2229797T3 - BIDIRECTIONAL REACTIVE STRIPS OF SIDE FLOW AND CORRESPONDING PROCEDURE. - Google Patents

Abstract

A test strip adapted to receive a sample and detect an analyte therein is provided. The test strip comprises a sample addition zone to which a sample may be added; an absorbent zone proximal to the sample addition zone; one or more test zones distal to the sample addition zone, at least one of the test zones including a first analyte binding agent immobilized therein which is capable of binding to the analyte to be detected; and a terminal sample flow zone distal to the one or more test zones, the absorbent zone being positioned relative to the sample addition zone and having an absorption capacity relative to the other zones of the test strip such that a distal diffusion front of a sample added to the sample addition zone diffuses from the sample addition zone to a distal diffusion point within the terminal sample flow zone and then reverses direction and diffuses proximal relative to the one or more test zones.

Description

Bidirectional lateral flow test strips and corresponding procedure.

Field of the Invention

The present invention relates to strips lateral flow reagents and operating procedures for such lateral flow test strips.

Summary of the invention

A test strip is provided that is adapted to receive a sample and detect an analyte in it. According to an embodiment of the invention, the test strip comprises: a sample addition zone to which a sample; an absorbent zone near the zone of addition of samples; one or more test zones distant from the zone of addition, comprising at least one of the test zones a first analyte binding agent, immobilized in said area, which is capable of agglutinating with the analyte to be detected and a terminal sample flow zone to one or more of the zones of test, the absorbent zone being located relative to the zone of sample addition and absorption capacity relative to other areas of the test strip, such that a front of distal diffusion of a sample, added to the zone of addition of samples, diffuse from the zone of addition of samples to the point of distal diffusion within the terminal sample flow zone, then reversing his address and spreading proximally with respect to one or more of the test areas.

In another embodiment, the test strip comprises furthermore an addition zone of conjugate buffer solution, distal to the terminal sample flow zone, to which the conjugate buffer solution.

According to the realization of the test strip above, the zone of addition of conjugate buffer solution can be place relative to the test areas, so that the solution conjugate buffer, added to the buffer solution addition zone conjugate at the same time as the sample is added to the zone of adding samples, reach the distal diffusion point after that the distal diffusion front of the sample has spread to the point of distal diffusion and begun to spread in a next address. The addition zone of conjugate buffer solution it can also be relative to the test areas, thereby that the buffer solution, added to the solution addition zone conjugate buffer at the same time as the sample is added to the zone Sample addition, reach the test areas after the distal diffusion front of the sample is diffused proximally in relationship with the test areas. The solution addition zone conjugate buffer can also be located relative to the areas of test, so that the conjugate buffer solution can be add to the test strip before the sample and, nevertheless, I reached the distal diffusion point after the front of distal diffusion of the sample has been disseminated to the area of distal diffusion, reversed its direction and started to spread in a proximal direction.

According to any of the previous embodiments of the test strip, the test strip may comprise 1, 2, 3 or more test zones with one or more control binder agents, immobilized in it. In one embodiment, the test strip comprises at least a first control zone with an agent control binder immobilized in it. Optionally, the test zones further comprise a second control zone with the same immobilized control binding agent as the first control zone, the first control zone containing a different amount of the control binder than It contains the second control zone.

Also, according to any of the embodiments above the test strip, a second binding agent of analytes that is able to agglutinate with the analyte and spread to One or more test zones can be understood in the test strip. Alternatively, the second analyte binding agent is can provide the test strip through the buffer solution conjugated The second analyte binding agent can be bind with components of the sample other than analyte. How alternatively, the second analyte binding agent may be a agent that does not bind with the sample components other than analyte.

In order to facilitate detection, the second analyte binding agent is preferably labeled with a detectable marker. As described here, it can be used any of a wide range of known detectable markers In this technique. In a preferred embodiment, the second agent analyte binder is added to a particle that is capable of spread to one or more test areas. The particle can serve as the detectable marker or can, by itself, be labeled with a detectable marker.

A procedure is also provided for Detect an analyte in a sample. In an embodiment of the invention, the method comprises depositing a sample in a sample addition zone of a test strip, comprising said test strip an absorbent zone close to the zone of sample addition and absorption capacity relative to other areas of the test strip, which makes a diffusion front distal sample (a) diffuses in a distal direction to one or more test zones, comprising at least one of said zones a first analyte binding agent immobilized therein, which agglutinates with the analyte in the sample, (b) diffuses to an area of distal terminal sample flow relative to one or more areas of test, change direction and (c) diffuse to a nearby position to one or more test areas; adding a buffer solution conjugated to the test strip in a distal position with respect to the Terminal sample flow zone, causing delivery of the conjugate buffer solution than a second binding agent of analytes spread, proximally, beyond the area of terminal sample flow to one or more of the test zones, after the distal diffusion front of the sample is diffuse proximal to one or more of the test areas, agglutinating the second analyte binding agent to the immobilized analyte in the test zones and detecting the second binding agent of analytes immobilized in the test areas.

Depending on the procedure, you can add a buffer solution conjugated to the test strip at the same time as the Sample is added to the test strip, before the sample is add to the test strip or after the sample is added to the test strip When the sample is added to the test strip relative to the conjugate buffer solution depends on the time necessary for the sample to reach the sample flow zone terminal which, in turn, depends on the flow design of the strip reactive

Also, according to the procedure, the second analyte binding agent may be contained in the strip reactive when the conjugate buffer solution is provided, causing the delivery of the conjugate buffer solution the diffusion of second analyte binding agent. As an alternative, the second analyte binding agent is contained in the strip reactive proximal to where the buffer solution is provided conjugate, which makes the delivery of the buffer solution conjugate generate the diffusion of the second binding agent of analytes Delivery of the buffer solution conjugated to the strip reactive may also comprise the addition of the second agent analyte binder to the strip, inside the buffer solution conjugated

According to the previous procedure, the zones of assay may comprise a first control zone with an agent control binder immobilized in it, causing the delivery of the conjugate buffer solution that a control agent diffuses, proximally, beyond the sample flow zone terminal, to the first control zone and there the agent is bound immobilized control binder. As an alternative, the areas of test can comprise first and second control zones, each one of which comprises a different amount of an agent immobilized control binder, causing delivery of the conjugate buffer solution that a control agent diffuses from proximally, beyond the terminal sample area, to the first and second control zones and bind the agent control binder there immobilized.

Also according to the previous procedure, the detection of the second analyte binding agent can be facilitated by labeling the second binding agent of analytes with a detectable marker, including detection of the second analyte binding agent the detection of marker. The second analyte binding agent can be added to a particle. The detection of the second binding agent of Analytes can comprise particle detection.

According to any of the previous embodiments, the sample delivered to the test strip is preferably within a predetermined range of volumes for which process is He has designed the test strip. The default volume range usually between 10 and 250 µl, preferably between 20 and 100 µl and more preferably between 30 and 50 µl and even more preferably between 35 and 45 µl. When a sample Delivered to the test strip within the volume range By default, the terminal sample flow zone can be design to have a short length from a proximal limb to a distal limb. For example, when a sample is delivered to the test strip within a range of approximately 35 to 45 \ mul, the terminal sample flow zone may have a length, from a proximal limb to a distal limb, of between about 1 to 25 mm, more preferably between 2 and 15 mm and even more preferably between 3 and 10 mm.

In addition, according to any of the embodiments above, the first non-binding analyte binding agent preferably components of the sample other than analyte. Among the types of molecules that can serve as the first agent analyte binder can be understood, without limitation, antibodies, engineering product proteins, peptides, haptens, lysates containing homogeneous mixtures of antigens with agglutination of analytes, ligands and receptors. In one embodiment In particular, the first analyte binding agent is a antibody or an antibody fragment.

Brief description of the drawings

Figure 1 illustrates a view from above to below an embodiment of a lateral flow test strip according to the present invention.

Figures 2A-2H illustrate a operating procedure for a lateral flow test strip, according to the present invention.

Figure 2A illustrates a sample that is being adding to the test strip.

Figure 2B illustrates the sample flowing inside of the test strip.

Figure 2C illustrates the test strip when the sample has circulated a distance inside the test strip, in the opposite direction to an absorbent zone within a zone of terminal sample flow.

Figure 2D illustrates the test strip where the Sample is circulating back to the absorbent area.

Figure 2E illustrates the addition of a solution buffer to the test strip.

Figure 2F illustrates the solution flow buffer inside the test strip towards the absorbent zone.

Figure 2G illustrates the solution flow buffer inside the test strip beyond the zone of test.

Figure 2H illustrates the solution flow buffer inside the test strip towards the absorbent zone

Figures 3A-3H illustrate a operating procedure for a lateral flow test strip according to the present invention.

Figure 3A illustrates a sample and a solution buffer that are being added to the test strip.

Figure 3B illustrates the sample and the solution buffer circulating inside the test strip.

Figure 3C illustrates the test strip when the sample has traveled a distance inside the test strip in the opposite direction to an absorbent zone within a flow zone of terminal sample.

Figure 3D illustrates the test strip where the Sample flows back into the absorbent area.

Figure 2E illustrates the buffer solution that follows circulating towards the sample flow.

Figure 3F illustrates the buffer solution that has circulated beyond the sample flow zone.

Figure 3G illustrates the solution flow buffer, inside the test strip, beyond the zone absorbent.

Figure 3H illustrates the solution flow buffer, inside the test strip, towards the absorbent zone.

Figure 4 illustrates the design of a strip reagent in which the sample addition zone is located adjacent to the zone of addition of the wash buffer solution.

Figures 5A-5C illustrate various designs of cartridges in which a strip can be placed reactive according to the present invention

Figure 5A illustrates a cartridge design adapted for the test strip illustrated in the Figures 2A-2H.

Figure 5B illustrates a cartridge design adapted for the test strip illustrated in the Figures 3A-3H, in which the sample addition zone is located at an extended distance from the zone of addition of the wash buffer solution, such that the sample and the Wash buffer solution can be added at the same time.

Figure 5C illustrates a cartridge design adapted for the test strip illustrated in Figure 4, in which the sample addition zone is adjacent to the area of addition of the wash buffer solution, the test area being located at an extended distance from the zone of addition of the wash buffer solution.

Figure 6A illustrates the general arrangement of an HP FLEXPACK ™ test strip manufactured by Abbott.

Figure 6B illustrates the operation of the strip reagent illustrated in Figure 6A.

Figure 7 illustrates a side sectional view of the lateral flow test strip illustrated in Figure 1.

Figure 8 illustrates the results of an assay Herpes 2 ReLIA ™ stop flow performed in the Example 2.

Figure 9 illustrates the results of an assay Herpes 2 ReLIA ™ stop flow performed in the Example 3.

Figure 10 illustrates the results of a ReLIA ™ stop flow Helicobacter pylori assay, performed in Example 4.

Description of preferred embodiments

The present invention relates to a strip lateral flow reactive that is able to make a part of a sample added to the test strip, flow from an area in the that the sample is added through one or more test zones towards a terminal sample flow zone and then with Independence of any user intervention, reverses the direction and flows backwards through one or more test areas to the sample addition zone. Fixed assets in at least one from the test zones there is a first binding agent of analytes that is able to agglutinate with an analyte in the sample for which the test strip is designed to detect it. Making a part of the sample circulate through one or more areas  test and then independently have a reflux in the direction of the sample addition zone, is eliminated the need to wash one or more of the test areas before come into contact with one or more test areas with a second analyte binding agent. It also eliminates the need for time when the second analyte binding agent is made spread to one or more test areas. As will be examined here with More details, the characteristics of self-washing and self-tightening of the test strips, according to the present invention, provide several significant advantages over previous strips reactive

The characteristics of autowash and self-timed test strips, according to the present invention, are achieved by adjusting the position of a absorbent zone relative to the sample addition zone, of such so that when a sample volume (within a range default sample volumes for said test strip) is added to the test strip, the diffusion front of the sample is expands through one or more test zones to an area of terminal sample flow. When the sample reaches the area of terminal sample flow, the absorbent properties of the area absorbent causes the sample to be dragged back through from the test zones to the sample addition zone and by last, to the absorbent zone.

Figure 1 illustrates a view of up-down one embodiment of a strip lateral flow reagent 100 according to the present invention. Is according illustrates, test strip 100 has proximal and distal extremities 102, 104, respectively, and can be divided into several zones different. The test strip comprises an addition zone of sample 106 where a sample can be added to the test strip 100. An absorbent zone 108 is located close to the area of sample addition 106. Distances from the sample addition zone 106 is one or more test zones 110, 112, 114. The strip reactive 100 also comprises a sample flow zone terminal 116, distal to one or more of test zones 110, 112, 114. Each of the above areas is in communication from diffusion of fluid to each other.

As illustrated, the test strip comprises also an addition zone of conjugate buffer solution 118, distal with the sample flow zone terminal 116. The zone of addition of conjugate buffer 118 may be an area in the that the conjugate buffer solution can be added to the test strip. Alternatively, the zone of addition of conjugate buffer solution 118 may simply correspond to an area whose solution conjugate buffer diffuses from a more distal point in the strip reactive

It should be noted that the configuration of the test strip illustrated in Figure 1 is linear in design. Without However, nonlinear configurations, such as configuration illustrated in Figure 4, are also provided for the strips reagents according to the present invention.

Figures 2A-2H illustrate a operating procedure of a lateral flow test strip, as shown in Figure 1. Before performing an assay using a test strip according to the present invention, is obtained a sample of fluid that is considered to contain the analyte that It will be detected. The sample may comprise any fluid that moisten the test strip and have a viscosity that is enough to allow displacement of the sample through of the test strip. In a preferred embodiment, the sample is an aqueous solution (such as a body fluid).

Figure 2A illustrates sample 120 that is added to a sample addition zone 106 of test strip 100. It must Note that the test strip is designed for use with a sample that has a volume within a particular range. More specifically, the delivery of a sample within the range default causes the sample to diffuse, distally, more beyond the test zones, towards the sample flow zone terminal 116, but not beyond the sample flow zone terminal 116 (as illustrated in Figure 2D).

As illustrated in Figure 2B, sample 120 begins to diffuse, both proximally and distally, through the test strip after being added to the test strip. As illustrated in Figure 2C, the distal front 124 of the sample 120 diffuses through one or more of the test zones 110, 112, 114, into the terminal sample flow zone 116. As illustrated in Figure 2D, the distal front 124 of the sample 120 extends, finally, to a point within the sample flow zone
terminal 116.

When the sample volume added to the strip reactive, within a predetermined range of volumes for the that the test strip is designed, the distal front 124 of the Sample 120 reaches a distal diffusion point corresponding to a maximum distal flow point, in a place within the zone of terminal sample flow 116. At this point, as illustrated in the Figure 2E, the capillary action by the absorbent zone 108 directs the shows, proximally, towards the absorbent zone 108. Tailored that the sample is carried towards the absorbent zone 108, the front distal 124 of the sample recedes proximally.

As illustrated in the Figures 2A-2D, a feature of the present invention It is the control of where and how the sample flows into the strip reactive The sample delivered to the test strip is found, preferably, within a predetermined range of volumes for which process the test strip was designed. The range of default volumes are preferably between 10 and 250 µl, preferably between 20 and 100 µl, more preferably between 30 and 50 µl, and more preferably still, between 35 and 45 µl. When a sample is delivered to a strip reactive within these ranges, the sample flow stops within the terminal sample flow zone.

The terminal sample flow zone can be design to have a short length from a proximal limb to a distal limb. For example, when a sample is delivered to a test strip within a range of 35 to 45 µl, the zone of sample terminal flow can have a length, from a limb proximal to a distal limb, between 1 and 25 mm, more  preferably between 2 and 15 and even more preferably, between 3 and 10 mm.

Located within one of the test areas (by example, zone 110), there is a first analyte binding agent which binds to an analyte in the sample for which the strip Reactive is planned for detection. The analyte present in the part of the sample that flows through the test areas is immobilizes in the test zone 110 by the first binding agent of analytes.

Figure 2E illustrates the addition of a solution buffer conjugated 122 to the test strip, in the zone of addition of 118 conjugate buffer solution, after the sample has Arrived at the terminal sample flow zone. Buffer solution conjugate 122 may contain one or more different second agents analyte binders that can bind the analyte and allow that is immobilized in the test areas to be detected. Be notes that the zone of addition of conjugate buffer solution 118 may optionally comprise one or more second agents analyte binders used to detect the analyte immobilized. In such case, the addition of the buffer solution conjugate 122 serves to initiate the diffusion of one or more seconds analyte binding agents across the areas of test.

As illustrated in Figures 2F and 2G, the conjugate buffer solution 122 flows, proximally, through the test strip in the direction of the absorbent zone 108, making thereby that one or more second analyte binding agents are move through test zones 110, 112, 114 and are agglutinate with the immobilized analyte.

As illustrated in Figure 2H, the action capillary through the absorbent zone 108 causes the sample 120 to diffuse into the absorbent zone 108. Meanwhile, the conjugate buffer solution 122 continues to spread, so proximally, through test zones 110, 112, 114 and within the absorbent zone 108. Any of the second agents analyte binders that were not immobilized in the areas test 110, 112 and 114, move with the buffer solution conjugate 122 towards the absorbent zone 108.

With regard to the embodiment illustrated in Figures 2A-2H, it is noted that the solution conjugate buffer 122 should be added to the test strip after that sample 120 has reached test zones 110, 112, 114 and, preferably, once the sample has reached the area of sample flow terminal 118 and has begun to spread towards back towards the absorbent zone 108. This allows the part of sample 120, which flows through the test areas, come into contact with the first binding agents of analytes, in the test areas, without the presence of the solution conjugate buffer.

Figures 3A-3H illustrate a alternative test strip design and operation procedure for the test strip. In this embodiment, the sample and the Conjugated buffer solution are added at the same time. With the purpose of that the sample and the conjugate buffer solution be added thereto time, it is necessary that the conjugate buffer solution reaches the test zones 210, 212, 214 after the sample has entered in contact with the test areas. It is preferred that the solution conjugate buffer reaches the test areas after the sample has begun to spread backwards through the test zones in the direction of the absorbent zone 208.

The delay with which the conjugate buffer solution it reaches the test areas, in this embodiment, it is achieved creating a greater distance between the solution addition zone conjugate buffer 218 and the terminal sample flow zone 216, in comparison with the design of the test strip illustrated in the Figures 2A-2H. As an alternative, it can be used a material that causes the conjugate buffer solution to diffuse from slower mode.

Figure 3A illustrates a sample 220 that is added to a sample addition zone 206 in the test strip 200. Meanwhile, a conjugate buffer solution 222 is added to the zone of addition of conjugate buffer solution 218, approximately at the same time that the sample is added to the test strip.

As illustrated in Figure 3B, sample 220 begins to spread, both proximally and distally, within of the test strip, once added to said test strip. Meanwhile, conjugate buffer solution 222 diffuses also, proximally and distally, within the test strip.

As illustrated in Figure 3C, the front distal 224 of sample 220 is diffused through one or more zones test 210, 212, 214 towards the terminal sample flow zone 216. Meanwhile, conjugate buffer solution 222 follows spreading, proximally, within the test strip towards The test areas.

As illustrated in Figure 3D, the front distal 224 of sample 220 finally extends to a point within the sample sample flow zone 216. At the time where the sample is in the terminal sample flow zone 216, the conjugate buffer solution 222 has not yet reached that zone.

As illustrated in Figure 3E, the action capillary through the absorbent zone 208 directs the sample, so proximal, towards the absorbent zone 208. As the sample is directed to the absorbent zone 208, the distal front 224 of the Sample flows proximally. Located within one of the zones test (for example, test zone 210) there is a first agent analyte binder that agglutinates the analyte in the sample, to whose detection was designed the test strip. The analyte, present in the part of the sample that flows through the areas of test, is immobilized in test zone 210 by the first agent analyte binder.

Figure 3F illustrates the conjugate buffer solution 222 that reaches the test areas. As you can see, in the at which point buffer solution 222 reaches the test areas, the distal front 224 of the sample has already circulated, so proximal, from the terminal sample flow zone 216 and the zones test 210, 212, 214.

As illustrated in Figures 3G and 3H, the capillary action by the absorbent zone 208 causes the sample to remove towards absorbent zone 208. Meanwhile, the solution buffer 222 continues to spread, proximally, through the test zones 210, 212, 214 and towards the absorbent zone 208. Any of one or more second analyte binding agents, that were not immobilized in test zones 210, 212, 214, they are transported with conjugate buffer solution 222 towards the area absorbent 208.

The conjugate buffer solution 222, added to the test strip, may contain one or more second agents analyte binders that can be agglutinated with the analyte and allow the detection of immobilized analyte in the areas of test. Alternatively, the test strip may comprise a zone  conjugated, distal to the terminal sample flow zone 216, which It contains one or more second analyte binding agents. The addition zone of conjugate buffer 218 can also Serve as a conjugate area. When one or more second agents analyte binders are preloaded on the strip reactive, the conjugate buffer solution 222 serves to initiate the diffusion of one or more second analyte binding agents to through the test zones towards the absorbent zone.

As illustrated in the Figures 3A-3H, the conjugate buffer solution can be added to the test strip before the sample reaches the areas of test designating the diffusion path of the test strip, of such that the conjugate buffer solution does not reach the areas of test until after the sample has been disseminated since test areas It should be noted that the dissemination of the solution conjugate buffer in test areas can be delayed enough for the conjugate buffer solution to be added to the test strip before adding the sample to the test strip.

Figure 4 illustrates an alternative design of the test strip for a lateral flow test strip according to the present invention The operation of a test strip is similar to the operation described in Figures 3A-3H, Los same reference numbers are used in Figure 4 as in the Figures 3A-3H. As illustrated in Figure 4, the Sample addition zone 206 is located adjacent to the zone of addition of conjugate buffer solution 218. This allows a design of more compact test strip, while allowing the sample and conjugate buffer solution are added simultaneously.

A feature of the strip design reactive, illustrated in Figure 4, is that the sample and the solution Conjugated buffer are added to the same end of the test strip. In addition, it is noted that test zones 210, 212, 214 are located towards an opposite end of the zones of addition of the sample and conjugate buffer solution 206, 218. This makes it is possible that the test areas are within the range of a Sample reader device while adding zones Sample and conjugate buffer solution are out of reach of the sample reader. This, in turn, allows the sample and the conjugate buffer solution is added to the test strip, while The test strip is in a test strip reader.

Figures 5A-5C illustrate various designs of cartridges in which you can place the test strips according to the present invention. In each design of cartridge, the cartridge comprises a sample addition hole 240 adjacent to the sample addition zone 206 of the strip reactive The cartridge also comprises an orifice for adding conjugate buffer solution 242 adjacent to the zone of addition of 218 conjugate buffer solution of the test strip. The cartridge it also includes a test window 244 adjacent to the zones test 210, 212, 214 of the test strip.

Figure 5A illustrates a cartridge design adapted for the test strip illustrated in the Figures 2A-2H. Figure 5B illustrates a cartridge design adapted for the test strip illustrated in the Figures 3A-3H, where the sample addition zone is located at an extended distance from the zone of addition of conjugate buffer solution, such that the sample and the solution conjugate buffer can be added to the test strip, in about the same time. Figure 5C illustrates a design of cartridge adapted for the test strip illustrated in Figure 4, where the sample addition zone is located adjacent to the buffer solution addition zone, the test zone being located at an extended distance from the zone of addition of buffer solution

It should be noted, regarding the Figures 2-4, which a characteristic of the test strips of the present invention is the inherent capacity of the strip test to expose test zones in the test strip to a part of the sample over a period of time and then make the sample diffuse away from the test areas, before the conjugate buffer solution reaches the areas of test. This feature is made possible by adapting (1) the adjustment of the position of the absorbent zone relative to the zone of sample addition with (2) the absorbent capacity of the strip reactive, between the sample addition zone and the flow zone of terminal sample, and (3) the volume of the sample to be deliver to the test strip. If too much sample is delivered, the Sample will not spread beyond the sample flow zone terminal. If too little sample is delivered, the sample will not be disseminated  far enough on the test strip to reach the zones of testing.

The ability of the test strip to expose the test areas to the sample for a limited period of time and to then have the sample removed from the areas of test, gives a timing independence to the strip reactive, which increases the accuracy of the test strip and It facilitates its use. For example, as illustrated in Example 2, the Test results do not depend on when the solution Conjugate buffer is added to the sample. As a result, the strips reagents do not need to be thoroughly controlled with respect to when the conjugate buffer solution must be added. In this regard, the time window, since the sample is added to when the Conjugated buffer solution should be added to the test strip, eliminated by the present invention.

The dynamics of using the volume of the Sample delivered to the test strip, to control how the Sample is diffused inside the test strip, it will now be illustrated referring to Figure 1. As stated above, Figure 1 illustrates a test strip that has proximal and distal extremities 102, 104, respectively, and Divide into several distinct zones. The test strip comprises a sample addition zone 106 where the sample is added to the test strip. An absorbent zone 108 is located close to the sample addition zone 106. A test zone 110 is located distal to the sample addition zone 106. A flow zone of terminal sample is located distal to test zone 110. A zone of addition of conjugate buffer 118 is located distal with the sample sample flow zone 116.

By way of illustration, the area of test 110 comprises a first analyte binding agent and the conjugate buffer solution addition zone 118 comprises a second analyte binding agent labeled with a marker detectable It is also assumed that the test strip is designed such that a sample volume of 30 µl will cause the Sample is disseminated up to a maximum of test zone 110. Meanwhile, a sample volume of 50 µl will cause the sample is diffused to the distal end of the flow zone of Sample terminal 116.

If a sample is delivered to the test strip within the range of volumes from 30 to 50 µl, the distal front of the sample will be disseminated beyond test zone 110. The distal advance of the sample will stop within the area of terminal sample flow 116. The analyte in the sample part which reaches test zone 110 will be agglutinated with the first antibody and will be immobilized in test zone 110. Other Sample components do not bind with the first antibody, since the first antibody is selective for the analyte. TO then the sample flows backward in the direction proximal to the absorbent zone 108 beyond the test zone 110. When the conjugate buffer solution is added, it causes the second analyte binding agent is diffused through the test zone 110 where the second analyte binding agent binds with the immobilized analyte in test zone 110. Since the sample diffuses away from test zone 110  before the buffer solution reaches test zone 110, no agents in the sample are present that could otherwise agglutinate with the second analyte binding agent. How result, the analyte to be detected in the sample does not have to compete with other agents in the sample, to be able to agglutinate with the second analyte binding agent.

If a sample volume less than 30 is delivered mul (for example, 25 mul) to the test strip, the sample does not never diffuses to test zone 110. As a result, none of the analytes in the sample reaches test zone 110 and binds with the first antibody. When the solution is added buffer, the second analyte binding agent is transported with diffusion of the conjugate buffer solution and crosses the area of test 110 without being immobilized, since no analyte is present in the test zone.

If the volume of sample delivered is greater than 50 µl (for example, 55 µl), the sample will spread more beyond test zone 110 and beyond the flow zone of terminal sample 116 towards the buffer solution addition zone conjugated Some of the analytes will agglutinate with the first analyte binding agent in test zone 110 and will remain immobilized. Meanwhile, some analytes will agglutinate with the second analyte binding agent in the zone of addition of conjugate buffer solution 118, before flowing backwards and agglutinate with the first analyte binding agent in the area Test 110. The analytes, which are agglutinated with two copies of the second analyte binding agent, it is unlikely that agglutinate with the first analyte binding agent, due to a steric hindrance. In addition, it may be more difficult for it to clump together the analyte with the first analyte binding agent once the analyte has already bound with the second binding agent of analytes. As a result, the sensitivity of the test strip may be reduced if the analyte binds with the second analyte binding agent, before agglutinating with the first binding agent. Other components of the part of the sample that reaches the zone of addition of conjugate buffer 118 they can also bind with the second binding agent of analytes These other components will compete with the analyte to agglutinate with the second analyte binding agent.

Controlling the volume of the sample delivered and therefore (1) exposing the analyte to the first binding agent of analytes before exposing the immobilized analyte to the second binding agent and (2) not exposing the second agents analyte binders before they are exposed to others sample components, non-specific agglutination is reduced, which greatly improves the sensitivity of the trial and the analyte detection accuracy.

As described above, two advantages of The test strips of the present invention are their properties of self-washing and self-cleaning. In order to explain the importance of these properties, a comparison with the HP FLEXPACK ™ test strip, manufactured by Abbot, which is illustrated in Figures 6A and 6B.

Figure 6A illustrates the configuration of the strip reactive As illustrated, the test strip comprises two separate sections 310, 312 that are joined together by a hinge 314. Section 310, on the right, comprises a strip reagent 316 comprising a sample addition zone 318, a test zone 319, a limit section 320 and a pad of Conjugated buffer solution transfer 322. Section 312, a on the left, it comprises an absorbent pad 324 that is located opposite the sample addition zone 318, a 326 conjugate buffer solution addition pad that is located opposite the buffer transfer pad  conjugate 322 and a test window 328 located in position opposite test zone 319. Opposite positions of the absorbent pad 324, the solution addition pad conjugate buffer 326 and test window 328 allow the absorbent pad 324 comes into contact with the addition zone of sample 318 and that buffer buffer addition pad conjugate 326 comes into contact with the transfer pad of conjugate buffer 322, when the first and second, 310 and 312, get in touch with each other. In addition, the area of test 319 can be seen through test window 328 when the first and second sections, 310, 312, are placed in contact with each other.

Figure 6B illustrates the operation of the strip reagent illustrated in Figure 6A. As illustrated, the solution 330 conjugate buffer is added to the buffer solution pad 326 conjugate. The 326 conjugate buffer pad comprises a second analyte binding agent (for example, an antibody) capable of agglutinating an analyte in the sample that is going to detect. The second analyte binding agent is labeled with a detectable marker that allows the display of the second binding agent. The second binding agent is not specific for the analyte and, therefore, can be agglutinated with others components in the sample.

A sample 332 is then taken and added to the sample addition zone 318. Once added, the sample is diffuses through test strip 316, from the addition zone of sample 318 through test zone 319. The test zone 319 comprises a first immobilized analyte binding agent (for example, an antibody) that binds selectively, with an analyte in the sample for whose detection the test strip When the sample crosses test zone 319, the analyte in the sample binds with the first agent analyte binder and is immobilized in the test zone 319.

When the broadcast front of the sample arrives to limit line 320, the user is supposed to put together the first and second sections, 310 312. The meeting of the sections first and second, 310, 312, makes the absorbent pad 326 direct the sample back into the sample addition zone 318, Meanwhile, the conjugate buffer solution is transferred to the  322 conjugate buffer solution transfer pad from the addition pad of conjugate buffer 320. The conjugate buffer solution diffuses from pad 322 to through test zone 319. The second binding agent of analytes, which was stored in the solution addition zone conjugate buffer 318, diffuse with the conjugate buffer solution and comes into contact with the immobilized analyte in the zone of addition test 319. The observation of the visually detectable marker in the second analyte binding agent, once immobilized in Zone 319, is used to detect the analyte.

According to the previous description of the operation of the HP FLEXPACK ™ test strip, you need to determine when the Sample reaches limit line 320 before making the solution conjugate buffer is transferred from the zone of addition of buffer solution 318 to the buffer pad addition conjugate 320 and start flowing into test zone 319. The affirmative stage of contacting the sample addition zone 318 with absorbent pad 324, in order to have the sample removed from the zone of Test 319. The design of the test strips herein invention, for example those illustrated in the Figures 2-4, eliminates the need to control the strip reagent to determine when the extraction of the Sample from the test area. In addition, since the sample is automatically removed, you do not need to carefully control the test strip with respect to when to add the buffer solution conjugated Instead, as illustrated in Example 2, the test results, using the test strips of the present invention, do not depend on when the buffer solution conjugate reaches the test areas, after the sample is disseminate from the test areas.

The lateral flow tests, according to the invention, They can be used in a wide range of applications. For example, the tests can be used to apply to the study of diseases humans, such as infectious diseases, or any other human diseases that involve recognizable epitopes (by example, cancer, AIDS, episodes and cardiovascular pathologies). The tests can also be used in veterinary applications, food, agricultural or chemical products testing of synthesis. The lateral flow tests, according to the invention, are can be used in numerous ways, including the use of a test apparatus of the lateral flow test, such as that of the patent application serial number 09 / 199.255, filed on 23 November 1998, which is incorporated here for the purpose of reference. In a preferred embodiment, the test apparatus of the lateral flow test comprises a ReLIA ™ test apparatus, available through PraxSys BioSystems (San Ramon, CA).

1. Construction of test strips according to the present invention

The procedures and materials for construction of the test strips, according to the present invention, They will now be discussed in more detail. It should be noted that the Particular construction of the test strips can be diverse, depending on the particular test that is intended to be carried out with the test strip Variations in the way the test strips can be built, regardless of this example, it is intended that fall within the scope of the invention.

Figure 7 illustrates a fragmented view of the test strip of Figure 1. As illustrated in Figure 7, the test strip 100 may comprise a backing strip 402 that is extends along the test strip. A 404 membrane band it is located on the backing strip 402 and serves as a step of diffusion for the test strip. On the membrane band 404 is located an absorbent pad 408, within the zone absorbent 108 that is positioned toward a proximal limb of the test strip Also on the membrane band 404 is located a sample pad 406, distal to the absorbent pad 408. An adhesive 409 can be used to bond the pad of sample 406 to membrane band 404. One or more test zones, 410, 412, 414, can be formed in membrane band 404 distal to the absorbent pad 406. An addition pad of 416 conjugate buffer solution is located on the band of membrane 404, distal to test zones 410, 412, 414 and distal to the sample sample flow zone 116. A protective cover 418 is located above the test areas. To allow escape the trapped air bubbles between the fluid fronts, a gap is left between the test zones and the zones conjugates that are not covered by protective cover 418. The protective cover 418 can also be placed more widely over the membrane band 404, in order to protect other parts of the test strip.

The backing strip can be made of any stable non-porous material, which is strong enough to support the materials and bands that fit. Market Stall that many tests use water as a diffusion medium, the strip of Backrest is preferably almost waterproof. In a preferred embodiment, the backing strip is manufactured with a polymer film, more preferably a chloride film of polyvinyl.

The backing strip can be manufactured from any substance that has sufficient porosity to allow capillary action of fluid along its surface and through its interior. The membrane band must have sufficient porosity to allow the displacement of particles coated with antigens or antibodies. The membrane band must also be wettable by the fluid used in the sample containing the analyte to be detected (for example, hydrophilicity for aqueous fluids, hydrophobicity for organic solvents). The Hydrophobicity of a membrane can be modified to prepare the hydrophilic membrane for use with aqueous liquids, by processes such as those described in U.S. Pat. No. 4,340,482 or in U.S. Pat. No. 4,618,533 which refers to the transformation of a hydrophobic surface on a hydrophilic surface. Between examples of substances that can be used to form the band Membrane fits: cellulose, nitrocellulose, acetate cellulose, fiberglass, nylon, ion exchange membrane polyelectrolytes, acrylic / nylon copolymers and polyethersulfone. In a preferred embodiment, the membrane band is manufactured with nitrocellulose

The absorbent pad can be formed from of an absorbent substance, which can absorb the fluid Used as the sample and buffer solution. The capacity of Absorption of the absorbent pad must be sufficiently great for absorbing fluids that are delivered to the strip reactive Examples of substances suitable for use in a absorbent pad comprise cellulose and fiberglass.

Buffer solution addition pad It can be formed by any absorbent substance. Examples of substances that can be used, include cellulose, nitrate cellulose, cellulose acetate, fiberglass, nylon, membrane Ion exchange of polyelectrolyte, copolymer / acrylic nylon and polyethersulfones.

As discussed above, the pad of addition of conjugate buffer solution can serve as a conjugate pad and contains an agent labeled with a detectable marker, which is capable of agglutinating with the analyte that It will be detected in the sample. As an alternative, the test strip may comprise a conjugate pad separate from the buffer pad addition, containing an agent labeled with a detectable marker, which is capable of agglutinating with the analyte to be detected in the sample.

The protective cover may be formed of any material that is waterproof and is preferably translucent or transparent. The protective cover may have a single layer or multiple layers. Preferred materials for use in the protective cover comprise materials optically transmitters, such as polyamide, polyester, polyethylene, acrylic compounds, glass or the like. The cover protective may be transparent, or not, depending on the Detection procedure used. In a preferred embodiment, the protective cover is an optically polyester transparent.

2. Tests for use with test strips according to present invention

The test strips of the present invention They are intended for use with a wide range of lateral flow, involving two analyte binding agents, each of which can agglutinate an analyte that is going to detect. At least one of the binding agents must agglutinate, selectively, the analyte. More specifically, one of the binding agents should agglutinate the analyte and not Any other components of the sample.

As used herein, the term "analyte" refers to any component of a sample (for example, molecule, compound or aggregate) to be detected and, as an option, determined quantitatively by means of a test strip test. Examples of "analytes" include  include proteins, such as hormones and other proteins secreted, enzymes and surface proteins of cells; glycoproteins; peptides; small molecules; polysaccharides; antibodies (comprising monoclonal and polyclonal antibodies and its fragments); nucleic acids; drugs, toxins; virus or virus particles; parts of a cell wall and other compounds that have epitopes.

The first and second of the analyte binding agents can be any agents that can bind the analyte to be detected. A wide range of different types of molecules can be used as analyte binding agents, comprising, for example, antibodies, design proteins, peptides, haptens and lysates containing heterogeneous mixtures of antigens with analyte binding sites, P. Holliger et al , Trends in Biotechnology 13: 7-9 (1995); SM Chamow et al , 'Trends in Biotechnology 14: 52-60 (1996). If the analyte to be detected is a ligand, a receptor that binds the ligand and vice versa can be used. In a particular embodiment, the first and second analyte binding agents are antibodies that bind an immunogenic part of the analyte.

Note that at least one of the first or second analyte binding agent has to agglutinate the analyte and not agglutinate any of the other components of the sample that it will be analyzed, referred to here as an agent selective analyte binder. In one embodiment, the first analyte binding agent, which is immobilized in an area of assay, is a selective analyte binding agent and the second analyte binding agent, which is labeled with a marker detectable, it is capable of agglutinating, non-selectively, the analyte In another embodiment, the first binding agent of analytes, which is immobilized in a test zone, is capable of agglutinate, non-selectively, the analyte and the second agent analyte binder, which is labeled with a marker detectable, it is a selective analyte binding agent. In other realization, both the first and second agents analyte binders are selective binding agents of analytes

Examples of selective analyte binding agents may be mentioned antibodies (monoclonal, polyclonal and fragments thereof) that have a close binding affinity for only a particular type of biomolecule, such as a protein or a receptor. The detectable label, incorporated into the second analyte binding agent, can comprise a wide range of materials, as long as the label can be detected. Examples of detectable markers include, without limitation, particles, luminescent labels; colorimetric labels, fluorescent labels; chemical labels; enzymes; radioactive tags or radio frequency tags; metal colloids and chemiluminescent labels. Examples of the most frequently used detection methodologies include, without limitation, optical procedures, such as measurement of light scattering, simple reflectance, luminometer or photomultiplier tube; radioactivity (measured with a Geiger counter, etc.); electrical or dielectric conductivity (capacitance); electrochemical detection of released electroactive agents, such as indium, bismuth, gallium or tellurium ions, as described by Hayes et al (Analytical Chem. 66: 1860-1865 (1994)) or ferrocyanide as suggested by Roberts and Durst (Analytical Chem 67: 482-491 (1995)), wherein the ferrocyanide encapsulated within a liposome is released by the addition of a drop of detergent in the detection zone, with the subsequent electrochemical detection of the released ferrocyanide. Other conventional procedures may also be used, when deemed appropriate.

It may be desirable to test two or more analytes different using the same test strip. In such cases, you can it is advisable to use different detectable markers in the same test strip, where each detectable marker detects a different analyte For example, different detectable markers are they can incorporate different selective binding agents of analytes The different detectable markers can be different fluorescent agents, which emit fluorescence at Different wavelengths

When two or more different analytes are detected using the same test strip, they can optionally be formed separate test zones on the test strip for each analyte That is going to be detected. The same detectable marker can be used for all analytes. As an alternative, different detectable markers, such as the above, can be used for different analytes, in order to prevent an area from being confuse with another.

In a preferred embodiment, the marker Detectable is a particle. Examples of particles that can used include, without limitation, colloidal gold particles; colloidal sulfide particles; colloidal selenium particles; colloidal particles of barium sulfate; colloidal particles of Iron sulphate; metal iodate particles; particles of silver halide; silica particles; colloidal particles of metal oxide (hydrated); colloidal sulfide particles metal; colloidal particles of lead selenide; particles cadmium selenide colloidal; colloidal phosphate particles metal; colloidal particles of metal ferrite; any of the above colloidal particles coated with layers organic or inorganic; protein or peptide molecules; liposomes or latex particles of organic polymers, such as polystyrene latex beads.

A preferred class of particles is that of colloidal gold particles. The colloidal gold particles are they can get by any conventional procedure, such as the procedures set forth in G. Frens, 1973 "Nature Physical Science ", 241: 20 (1973). Alternative procedures may be those described in U.S. Pat. No. 5,578,577, No. 5,141,850; nº 4,775,636; No. 4,853,335; No. 4,859,612; No. 5,079,172: No. 5,202,267; No. 5,514,602; No. 5,616,467 and No. 5,681,775.

The particle size selection can depend on factors such as reagent stability of bulk colloid (sun) and its conjugates, performance and the integrity of the particle release of the pad conjugates as well as the speed and integrity of the reaction. Also, the surface area of the particles can influence steric hindrance between agglutinated molecular halves. He particle size can also be selected based on of the porosity of the membrane band. Preferably, the particles must be small enough to spread to membrane length due to the capillary action of the buffer solution conjugated

The particles can be labeled to facilitate its detection Examples of labels can be cited without limitation, luminescent labels, such colorimetric labels as dyes; fluorescent labels or chemical labels, such as electroactive agents (for example, ferrocyanides); enzymes; radioactive tags or radio frequency tags.

The number of particles present in the strip reactive may vary, depending on the size and composition of the particles, the composition of the test strip and the band of membrane as well as the level of sensitivity of the assay. The number of particles usually varies between about 1 x 10 9 and 1 x 10 13 particles, although less than 1 x 10 9 particles. In a preferred embodiment, the Particle number is about 1 x 10 11.

3. Control test zones

As illustrated in Figure 1, on the strip There may be a plurality of test zones 110, 112, 114. Each test zone is located in such a way that a analytical, automatic or semi-automatic instrument, or a reader human, can determine some results of the flow test side.

As stated above, immobilized in at least one of the test areas is a first analyte binding agent, which is capable of agglutinating with the analyte in the sample for whose detection the test strip is designed. In some embodiments, it may be advisable, to some of the other test areas, include one or more areas of control, in which one or more agents have been immobilized control binders. Control agents capable of agglutinating with the control binder they can be incorporated into the test strip in various positions or added to the test strip when the test is being carried out. The control agents are preferably labeled with a detectable marker, such as the above detectable markers, to facilitate the detection of the control agent that binds with the agent control binder immobilized in a control zone.

Control agents and binding agents control can be used in combination to perform a Wide range of control functions. For example, the pairs of control binders can be used to confirm if the sample and the conjugate buffer solution have been diffused properly inside the test strip. The pairs of control binders They are also usable as internal standards and allow analyte measurement results can be compared between Different test strips. This can be used to correct the variability between one strip and another. Such correction could be unfeasible with external controls that are based, for example, in a statistical sampling of the strips. In addition, the variations batch to batch and use to use between different test strips can be minimized using pairs of control binders. In addition, the effects of a non-specific binder, as will reveal later, they can also be reduced. All these Corrections are difficult to perform using external controls Out of the strips.

In this technique, a wide range of agents that can be used as members of the binder pair of control. For example, at least one element of the pair of Control binders can be a natural or designer protein. The pair of control binders can also be a pair of ligand-receptor. In addition, at least one element of the pair of control binders can be an antigen, another organic molecule or a hapten conjugated to a non-protein specific to the analyte object of interest. Descriptions of other suitable elements of pairs of control binders can found in US Patent No. 5,096,837 and include IgG, others immunoglobulins, bovine serum albumin (BSA), other albumin, casein and globulin.

Desirable characteristics for pairs of control binder agent-control agent, but not limited to stability as a whole, they are the ones that are missing of specificity for the analyte of interest, reproducibility and predictability of test behavior, molecular size and greed for agglutination with each other.

In a preferred embodiment, the elements of the pair of control binders do not agglutinate with anything that can be present in the test strip, for example, coming from the sample. In one embodiment, the control binder comprises an anti-dinitrophenol (anti-DNP) rabbit and control agent comprises a dinitrophenol conjugated to BSA (serum albumin bovine)

In a preferred embodiment of the invention, both the second analyte binding agent, which diffuses to along the test strip, as the control agent will incorporates a unique species of particles. The incorporation can be by non-specific absorption or by chemical procedures of traditional conjugates. As an alternative, a system of non-covalent agglutination, such as "biotin-avidin", or even an antibody specific for the second analyte binding agent, it can be use to incorporate the analyte binding agent to the particle. Bifunctional and multifunctional reagents can also used to couple the second binding agent of analytes and the control agent with the particle.

The number of the second binding agents of analytes and control agents incorporated into each particle are may vary, depending on what is most appropriate for each test. For example, two copies of the second binding agent of analytes and a copy of control agent can be incorporated into Each particle Alternatively, a copy of the second agent analyte binder and two copies of the control agent can be incorporated into each particle. Other variations on relations between the second analyte binding agent: agent Control: particle can be used depending on the test particular in which they will be used, these variations being intended to fall within the scope of the present invention.

In a preferred embodiment, the test strip It comprises more than one control zone and is used to create a calibration curve with respect to which a wide range of analyte measurement results. This embodiment is described in more detail in the patent application serial number 09 / 198.118, filed on November 23, 1998, which is incorporated here by reference. Make the strip reactive owning more than one control zone allows tests lateral flow have a wider dynamic range than conventional lateral flow tests. In realizations preferred, the test strips with 2, 3 or more control zones are use with a small-scale methodology, discussed below, which allows the graphical representation of quantities of pairs of control binders detected on the same scale on which reports the amounts of analytes detected.

In a preferred embodiment, the test strip it has at least one high control zone and at least one zone Low control The difference between the two zones usually consists in concentration The concentration of the control agent in the high control zone is greater than the concentration of the agent control in the low control zone. Therefore, the amount of pairs of control binders will be higher in the control zone high than in the control zone low. In embodiments in which the number of pairs of control binders, in an area of given control, can be represented graphically with the same scale as used to indicate the amount of analytes, you can draw a calibration curve through the values of the pairs of binders in the high and low control zones.

In other embodiments, more than two zones of Control may be present. This allows to generate a curve that best reflects any nonlinearities present in the trial between the amount of analyte detected and the measurement against which the quantity could be represented graphically, as explained more ahead. Although such linearity failures could affect, of somehow, to the trials that involve a relatively relationship linear, can be corrected to use multiple control zones. 2, 3 or more control zones can be used.

In another embodiment, a single control zone It may comprise more than one type of control agent. This can be employ in embodiments where there is more than one population of analyte binding agents and non-specific agents of analytes coupled to a detection agent. For example, when you want to test two or more analytes of interest with the same strip reactive, two populations of binding agents can be prepared of analytes and non-specific analyte agents coupled to a detection agent Different agents of detection for each population, thus allowing to establish a distinction between the results for the two different analytes that They are of interest. In such circumstances, it might be advisable use control zones that include different control agents or pairs of control binding agents.

The control zones can be placed in a wide range of locations within the group of zones of test. It is noted that the test areas can be placed in various places on the test strip, depending on the design of flow of the test strip compatible with the present invention.

The tests are performed using a test strip comprising one or more control zones as part of the zones of test in the same way as described with respect to the Figures 2A-2H, and 3A-3H. It is noted that the test strip, or conjugate buffer solution, comprises the agent of control that binds with the control binder immobilized, for example, in test zones 112 and 114 of the Figures 2A-2H and in test zones 212 and 214 of Figures 3A-3H. When the solution is added conjugate buffer, the control agent diffuses with the solution conjugate buffer and binds with the control binder immobilized in control areas.

The amounts of control agents immobilized in the control zones, they are detected together with the detection of quantities of the second analyte binding agent immobilized in the test area. As noted above, it is preferable  that the control agents and the second binding agent of analytes are labeled with a detectable marker, which facilitates its detection The amount of detectable marker in each zone of assay can be determined, quickly, by a wide range of techniques known in this technique, depending on the type of detectable markers that are being used. Common examples of detection techniques comprise optical procedures (light scattering, simple reflectance, luminometer or tube photomultiplier); radioactivity; electric conductivity; dielectric capacitance; electrochemical detection of agents electroactive released, as indicated above.

Once the amount of detectable markers in each test zone, these measurements can be used to detect and preferably quantify the amount of analytes present, preferably also by calibration of the test strip using the amounts of detectable markers in control zones. For example when high and low control zones are used, the amount of agent immobilized control in the high and low control zones can be use to quantify the amount of the second binding agent of analytes in relation to the high and low control zones. These Relative intensity measurements can be used, later, to more accurately determine the number of analyte copies present in the measurement volume.

An advantageous feature of using areas of Multiple control consists of the ability to create a scale relative for analyte measurements. Once they have quantified the amounts of detectable markers, these detectable marker amounts can be represented graphically, then with another measurement scale. By example, even when analyte measurement results can be measured based on an absolute measurement of the analyte, the results obtained may be more significant in others units, such as an intensity relative to that of an area of control, or control zones, mentioned here as Intensity Relative or IR. The results can also be expressed as the number of analyte copies present in the measurement volume. The graphic representation of the amount of analytes detected, in other measurement scales, is a preferred embodiment to inform of the results of the test according to the invention.

For example, the test results are They can graphically represent a relative scale. Using a relative scale, such as Relative Intensity (IR), for internal controls, the absolute values for the Detected analyte can be converted to IR values. In a preferred embodiment, an IR value can be assigned to a low control of 1 and a high control can be assigned an IR value of 3, even when the ratio of absolute values of these controls can be different. In a preferred embodiment, the ratio of values Absolutes can be at least about 5: 1, while the IR ratio can be around 3: 1. In doing so, the changes in the individual test strips that affect the measured absolute value will cause the standard curve to move up and down an axis of Y coordinates, but it will have a minor impact on the IR value plotted along the X axis. This systematically attenuates the variability in the result. informed, that is, it will manifest as a "gain negative".

For example, if there is a negative gain between the measured quantity of analytes and the quantity reported, so, the big changes in the amount of analyte measured will have a graphic correlation with the changes relatively small in the reported amount of analyte. Even if I don't change the underlying variability of the measured amount of analyte, the Procedure may be useful in certain circumstances. The negative gain effect attenuates part of the variability of the test and can be used to improve the reproducibility of reported test results compared to simple information of an absolute value of what has been measured.

In addition to reporting the results of essays, on a continuous scale, either directly as the amount of analyte detected, either indirectly as a scale measurement in which the quantity has been plotted of analyte detected, the tests according to the invention can be used in a "cut" type essay. If the detectable marker is detects in an area of analyte agglutination, the amount of detectable marker detected can be compared with respect to a cutting value A cutoff value is the value above which the test can be considered positive; that is, the analyte of interest is present in the fluid sample in some degree of statistical confidence Below the cut-off value, the test is usually considered not positive-the analyte of interest is not present or the lateral flow test according to the invention, He did not detect his presence. Although a cut can be established on the basis of a directly measured value, such as the amount of analyte detected, the results may be more significant if They are provided on an indirect or relative scale.

A lateral cutting flow test is more desirable since it increases the measurement separation between a Negative value and a positive value. A negative value is the value indicated on a continuous scale, in the case where the analyte of interest is not statistically present. On the contrary, a positive value is the value indicated on a continuous scale, in the If the analyte of interest is statistically present. TO as these values converge, the probability of be able to indicate, in statistical terms, the positive values and negatives separately.

A lateral cutting flow with greater precision in cutting. When there is less variation in the chosen cut, it is more likely that a positive value can be considered exactly a positive and a negative value be considered exactly a negative.

The test results can be represented on a "relative" scale, previously examined or on an "absolute" scale. The absolute scales are measured in real physical units, such as number of copies of analytes per milliliter of fluid. The measurement on the absolute scale It may be preferable for testing some diseases or conditions, such as cancer marker tests. In said preferable embodiments, the result can be expressed in units such as ng / ml. Consequently, control zones may have concentrations with assigned agent value of control. In an extension of the concept of relative measurement, it you can measure the density of the density values of reflectance (DR) of a series of concentration patterns in known analytes and can be calculated, as previously described, the intensities relative to the controls (values of GO). Then the IR values can be taken to a graph regarding the concentration in analytes to build a standard curve in which IR values are assigned values of concentration of the analyte of interest. The IR value of a sample it can then be read in this pattern curve of assigned value, which provides a result labeled in the desired units.

Where possible, it is desirable to employ an agent unique as, at the same time, the analyte binding agent and the agent of control. Compared to tests where the binding agent of analytes and the control agent are separate agents, the trials where a single agent is used provide one more Wide separation of population measurement negative samples and positive, along with greater precision in cutting.

Numerous circumstances can affect the absolute reactivity of lateral flow tests comprising, without limitation, variations derived from manufacturing, operator induced variations, induced variations for the environment and effects of the samples. With essays of Conventional lateral flow, any of these variations they can act to reduce or supposedly expand the reactivity of one band over the other, leading to possible negative results or false positives. Not controlling these or other variations may lead to significant inaccuracy, lack of reproducibility, lack of sensitivity and lack of specificity of the trials.

The lateral flow tests are also subject to various interferences that could affect the magnitude Agglutination absolute of the analyte binding agent or control agent for test areas. The facts influencers may include: 1) variability in the release of second analyte binding agent or control agent from a conjugate support, 2) variation from device to device in non-specific agglutination of the binder population of analytes for the test strip, 3) variability in the movement of the population of analyte binders across or along the test strip during the test due to variation in the size of the pores of the test strip or membrane band materials or  non-specific aggregation of the analyte binding agent. The variability of absolute agglutination measurements, due to these or other factors, it may, therefore, be inadmissibly high in conventional lateral flow tests.

These kinds of variability can be reduced using a single agent comprising the second agent analyte binder and control agent. Any part of the lateral flow test matrix that has been exposed to the agent control is more likely to have been exposed to the second agent analyte binder, compared to two trials conventional populations. Any mechanism that prevents or avoids the movement of the control agent along or through the lateral flow matrix is more likely to prevent or avoid the movement of the second analyte binding agent, in comparison with the trials of two conventional populations. In Third, the control agent can be chosen so that reduce the magnitude of the non-specific agglutination of the second analyte binding agent.

The reduction of non-specific agglutination may also occur due to modification of the profile of hydrophobicity / hydrophilicity of the detection matrix of analytes The reduction in the "self-association" of aggregation of the particles of the analyte detection matrix, which could prevent the movement of the matrix along the band, it can also achieved by selecting a control agent with adequate properties. A final advantage is that, due to the need to prepare less reagents, expenses can be reduced of manufacturing.

Multiple control zones, as here reveals, they offer several possible advantages in the practice of lateral flow tests. Control zones can be used additional to expand the dynamic range of the standard curve of test whether the curve is linear or nonlinear. Multiple zones of control can also be used to define whether an effect of High dose hitch or prozone is present in a given trial. If this effect is present, then it can be recommended to the user dilute the sample to determine, without ambiguity, the concentration of the analyte in question.

Examples 1. Construction of the test strip

In this example, the construction of a test strip having a design as illustrated in the Figures 1 and 7. Milipore SRHF nitrocellulose backed sheets (4.8 cm x 20 cm) (404 membrane band) were coated dispensing, in the longitudinal direction, an antigen band (zone of  test 410) and two control bands (test zones 412, 414) in 404 nitrocellulose using a Bio Dot dispensing platform XYZ3000 with Biojets operating at a frequency of 120 Hz, 20, 83 nl / drop and 0.5 µl / cm. The 404 nitrocellulose sheets were then dried for one hour at a temperature of 37 ° C in a forced air incubator, locked for fifteen minutes in a PBS solution containing 10 mg / ml BSA, 1% (by weight / volume) PEG 8000, 3% (by weight / volume) mannitol, 0.3% (by weight / volume) gelatin, 0.01% (by weight / volume) of sodium azide and 0.05% (in weight / volume) of sodium dodecyl sulfate and then dried during a additional time in a forced air incubator at a temperature of 37 ° C. The 404 coated nitrocellulose sheets were stored dried at room temperature in leaf bags metallic

Gelman 8980 fiberglass pads for use as 416 conjugate buffer pads were preblocked by immersion in a solution of PBS 10 mg / ml BSA, 1% (weight / volume) of Triton X-100, 2.5% (weight / volume) of sucrose and 0.3% (weight / volume) of polyvinylpyrrolidone K-30 and then a 2 hour drying in an incubator of forced air. A conjugate solution in PBS 10 mg / ml BSA, 1% (weight / volume) of Triton X-100, 2.5% (weight / volume) of sucrose and 0.3% (weight / volume) of polyvinylpyrrolidone was dispensed, longitudinally, on the pads of 416 preblocked conjugate buffer solution using a Bio Dot XYZ3000 dispensing platform with a unique Biojet operating at a frequency of 120 Hz and providing 104.17 nl / drop and 2.5 µl / cm. 416 conjugate buffer pads were coated with conjugate in patterns of two to six lines per cm with three coated patterns on each 3 cm pad x 10 cm Coated conjugate buffer pads 416 were dried under vacuum in 2 Torr for two hours at room temperature and then cut into three 1 cm sections x 20 cm each containing a pattern.

Test strips were prepared by fixing a sheet of nitrocellulose backed 404 of 4.8 cm x 20 cm, a pad of conjugated buffer solution coated 416 of 1 cm x 20 cm and a sheet 406 of Gelman Cytosep 1662 of 1.2 x 20 cm in a sheet of 0.02 '' thick 402 vinyl backing, 6 cm x 20 cm, Adhesive coated (precision die cut by G&L). One sample application pad 406 0.5 cm wide was fixed to nitrocellulose 404 using a double adhesive side 409. Strips 0.5 cm wide were cut from the blade assembled with a die cutting blade G&L accuracy. To assemble the test strip in a test cartridge (illustrated in Figure 5A), the strip was placed in the lower half of the support, an absorbent pad 408, 0.6 cm x 1.5 cm, was placed on top of the strip and the pins in the upper half of the stand were aligned with the holes in the bottom half and were pressed together Hermetically

2. Analysis of the addition time of buffer solution and volume dependence of test strips

The strips used in this example were constructed as described in Example 1. The strips are coated with 800 µg / ml anti-dinitrophenyl (anti-DNP) rabbit in the high control band (test zone 414), 200 µg / ml of rabbit anti-dinitrophenyl (Anti-DNP) in the low control band (test zone 412) and a solution of Herpes 2 gG2 antigen having an optical density, at 280 nm, of approximately 0.115 in test zone 410. The order of the bands inside the test strip was the most antigen band next to the conjugate buffer pad, zone of low control between the antigen band and the high control band further away from the conjugate buffer pad and more next to the absorbent pad.

Buffer solutions pads 416 preblocked conjugates were coated with conjugate of A-OMNI? protein [protein A / BSA-DNP (2 x / 0.5 X)] - 8.5 nm gold, mixing three volumes of the conjugate stock solution (OD 520 approximately 63) with a volume of PBS containing 40 mg / ml of BSA, 4% (weight / volume) of Triton X-100, 10% (weight / volume) of sucrose and 1.2% (weight / volume) of polyvinylpyrrolidone K-30 and 0.15 volumes of 20 x PBS The mixture was dispensed on the solution pads preblocked conjugate buffers as described in the Example one.

In rehearsal it was done by placing the cassette on the laboratory bench and then adding 32.5 µl, 35 µl, 40 µl, 45 µl or 50 µl of moderate positive sample 705145 from Herpes 2 to sample pad 406 through the Sample addition hole of the cassette. Then it added 125 µl of conjugate buffer solution (PBS, 10 mg / ml BSA, 0.1% sodium azide and 2 mM EDTA) to the addition pad of 416 conjugate buffer solution at 0, 5 or 15 minutes after the Sample will reach the sample flow zone terminal 116. A then the cassette containing the strip was placed in a ReLIA ™ machine set-up to operate and perform the ReLIA? assay reading for antibody detection for Herpes 2. The temperature of the strip was adjusted to 40 ° C and then the strips were read after 10 minutes

As can be seen from the results illustrated in Figure 8, the results of the Herpes 2 test of ReLIA? stop flow were independent of the added sample volume and incubation time, before initiation of reverse flow, for sample volumes from 35 to 45 µl and incubation times of up to fifteen minutes, being investigated the longest delay. Within this margin of volumes and incubation times, the result of the CV in the trial It was 12%.

3. Herpes test 2

The strips used in this example were coated with 800 µg / ml anti-dinitrophenyl rabbit (Anti-DNP) in the high control band, 200 µg / ml rabbit anti-dinitrophenyl (anti-DNP) in the low control band and a Herpes 2 gG2 antigen solution having a density optics, at 280 nm, of approximately 0.115 in the antigen band. The order of the bands was the closest antigen band to the conjugate buffer solution pad, low control zone between the antigen band and the low control bands and the band Low control farthest from the buffer pad conjugated and closer to the absorbent pad. They were coated nitrocellulose bands and test strips were prepared as described in Example 1.

Buffer pads were coated conjugate preblocked with protein conjugate A-OMNI? [Protein A / BSA-DNP (2X / 0.5X)] - 8.5 nm gold mixing three volumes of the conjugate stock solution (OD 520 approximately 63) with a PBS volume containing 40 mg / ml of BSA, 4% (weight / volume) of Triton X-100, 10% (weight / volume) of sucrose and 1.2% (weight / volume) of polyvinylpyrrolidone K-30 and 0.15 20 x PBS volumes. The mixture was dispensed on pads of preblocked conjugate buffer solutions as described in Example 1

In rehearsal it was done by placing the cassette on the laboratory bench and then adding 40 µl of the samples positive or negative Herpes 2, indicated in Figure 9, to the sample pad through the sample addition hole of the sample cassette. When the liquid front of the sample reached the end sample flow zone, the cassette containing the strip was placed in a ReLIA? machine configured for perform and read the ReLIA? assay for the detection of antibodies to Herpes 2. Initially 125 µl of conjugate buffer solution (PBS 10 mg / ml BSA, 0.1% sodium azide and 2 mM EDTA) to the conjugate buffer solution hole of the cassette. The temperature of the strip was adjusted to 40º C and the strips They were read after 10 minutes. The values of relative intensity (IR) of the samples were calculated according to a algorithm that assigns the control response under the test (reflectance density) with an IR value of 1, the response of high test control (reflectance density) with an IR value of 3 and zero response as an IR value of 0.

As illustrated in Figure 10, all positive samples of Herpes 2 provided intensity values relative (IR) of 0.58 or greater while all samples Herpes 2 negatives provided IR values of 0.01 or more low, showing that positive and negative populations are well separated in this essay.

4. Helicobacter pylori test

The strips used in this example were coated with 800 µg / ml of rabbit anti-dinitrophenyl (anti-DNP) in the high control band, 200 µg / ml of rabbit anti-dinitrophenyl (anti-DNP) in the low control band and a solution of Helicobacter pylori antigen having an optical density, at 280 nm, of approximately 1,157 in the antigen band. The order of the bands was the closest antigen band to the conjugate buffer pad, low control zone between the antigen band and the high control band and the high control band furthest from the conjugate buffer pad and closer to the absorbent pad. Nitrocellulose sheets were coated and test strips were prepared as described in Example 1.

Solution pads were coated conjugate buffers preblocked with protein conjugate A-OMNI? [Protein A / BSA-DNP (2 X / 0.5 X)] - 8.5 nm gold mixing three volumes of the conjugate stock solution (OD 520 approximately 63) with a PBS volume containing 40 mg / ml BSA, 4% (weight / volume) of Triton X-100, 10% (weight / volume) of sucrose and 1.2% (weight / volume) of polyvinylpyrrolidone K-30 and 0.15 20 x PBS volumes. The mixture was dispensed on pads of preblocked conjugate buffer solutions as described in Example 1.

The test was performed by placing the cassette on the laboratory bench and then adding 40 µl of the positive or negative samples of Helicobacter pylori , indicated in Figure 10, to the sample pad through the sample addition hole of the cassette of samples. When the liquid front of the sample reached the terminal sample flow zone, the cassette containing the strip was placed in a ReLIA? Machine configured to operate and read the ReLIA? Assay for the detection of antibodies for Helicobacter pylori . Initially, 125 µl of conjugate buffer solution (PBS, 10 mg / ml of BSA, 0.1% sodium azide and 2 mM EDTA) was added to the cassette of conjugate buffer solution of the cassette. The temperature of the strip was adjusted to 40 ° C and the strips were read after 10 minutes. The relative intensity (IR) values of the samples were calculated according to an algorithm that assigns to the low control response of the test (reflectance density) an IR value of 1, to the high control response of the test (reflectance density) an IR value of 3 and a zero response as an IR value of 0.

As illustrated in Figure 9, all positive samples of Helicobacter pylori provided relative intensity (IR) values of 1.64 or greater, while all negative samples of Helicobacter pylori provided IR values of 1.14 or less, thus demonstrating that the positive and negative populations are well separated in this trial.

It will be evident to experts in the matter that can be made several modifications and variations in the apparatus and procedures of the present invention without thereby departing from the scope of the invention. Therefore it is provided that the present invention covers its modifications and variations, provided they are within the scope of the attached claims and their equivalents. In addition, the Following examples have been included in order to illustrate the claimed invention and should not be construed as limiting of the scope of the claimed invention.

Claims (35)

1. Test strip (100) adapted to receive a shows and detects an analyte contained in it, which comprises: a sample addition zone (106) to which a sample (120) can be added; an absorbent zone (108) proximal to the area of sample addition; one or more test areas (110, 112, 114) distal from the sample addition zone, comprising less one of the test zones a first binding agent of immobilized analytes, which is capable of agglutinating the analyte that will detect, and a terminal sample flow zone (116) distal of one or more test zones, the absorbent zone being located relative to the sample addition zone and having a absorption capacity relative to the other areas of the strip reactive, such that a distal diffusion front (124) of a Sample added to the sample addition zone is subject to diffusion from the sample addition zone to a diffusion point distal within the terminal sample flow zone and at then be subject to proximal diffusion relative to one or more test areas 2. Test strip according to claim 1, in the that the test strip is adapted to receive a sample inside from a predetermined range of volumes. 3. Test strip according to claim 1, in the that the test strip is adapted to receive a sample inside of a predetermined volume range between about 10 and 250 \ mul. 4. Test strip according to claim 1, in the that the test strip is adapted to receive a sample inside from a predetermined range of volumes between approximately 20 and 100 \ mul. 5. Test strip according to claim 1, in the that the test strip is adapted to receive a sample inside from a predetermined range of volumes between approximately 30 and 50 \ mul. 6. Test strip according to any of the previous claims, wherein the sample flow zone terminal has a length from a limb proximal to a distal limb between approximately 3 and 10 mm. 7. Test strip according to any of the previous claims, wherein the first binding agent of analytes does not bind to components in the sample other than The analyte 8. Test strip according to any of the previous claims, wherein the first binding agent of analytes is selected from a group consisting of antibodies, designer proteins, peptides, haptens, lysates containing heterogeneous mixtures of antigens having sites analyte binders, ligands and receptors. 9. Test strip according to any of the claims 1 to 8, wherein the test zones comprise also at least a first control zone with an agent control binder immobilized in said area. 10. Test strip according to any of the claims 1 to 8, wherein the test zones comprise also a first control zone with a binding agent of immobilized control and a second control zone with the same immobilized control binder agent than the first zone of control, containing the first and second control zones a different amount of control binder immobilized. 11. Test strip according to any of the previous claims, wherein the test strip comprises also a distal zone with respect to the sample flow zone terminal comprising a second analyte binding agent, which is capable of agglutinating the analyte and spreading it to one or more areas of testing. 12. Test strip according to claim 11, in which the second analyte binding agent is capable of agglutinate components in the sample other than the analyte. 13. Test strip according to claim 11, in which the second analyte binding agent does not bind the components in the sample other than analyte. 14. Test strip according to any of the claims 1 to 13, wherein the second binding agent of Analytes is labeled with a detectable label. 15. Test strip according to any of the claims 1 to 14, wherein the second binding agent of analytes is attached to a particle that is able to spread to one or more test areas. 16. Test strip according to any of the previous claims, wherein the test strip comprises furthermore an addition zone of conjugate buffer solution (118) distal to the terminal sample flow zone at which it can add a conjugate buffer solution (122). 17. Test strip according to claim 16, in the extent to which it is subordinated to any of the claims 11 to 15, wherein the zone containing the second analyte binding agent is proximal to the zone of addition of conjugate buffer solution. 18. Test strip according to claim 16, in the extent to which it is subordinated to any of the claims 11 to 15, wherein the zone containing the second analyte binding agent is the solution addition zone conjugate buffer. 19. Test strip according to any of the claims 16 to 18, wherein the solution addition zone conjugate buffer is located in relation to the test areas, such that the conjugate buffer solution added to the zone of addition of conjugate buffer solution, while adding the sample to the sample addition zone, reach the point of distal diffusion after the distal diffusion front of the Sample has been disseminated to the distal diffusion point and the diffusion in a proximal direction. 20. Test strip according to any of the claims 16 to 19, wherein solution addition zone conjugate buffer is located relative to the test zone so that the conjugate buffer solution added to the zone of addition of conjugate buffer solution, at the same time the sample is added to the sample addition zone, reach the test zones after that the distal diffusion front diffuses proximally in relation to With the test area. 21. Procedure to detect an analyte in a Sample, comprising: deliver a sample (120) to an addition zone of samples (106) of a test strip (100), the strip comprising reactivates an absorbent zone (108) proximal to the zone of addition of samples and having an absorption capacity relative to other areas of the test strip that cause a front of distal diffusion (124) of the sample (a) is diffused in one direction distal to one or more test zones (110, 112, 114), comprising at least one of the test zones a first agent immobilized analyte binder that binds to the analyte in the sample, (b) broadcast to a terminal sample flow zone (116) distal to one or more test areas and (c) disseminate to a position proximal to one or more test areas; deliver a conjugate buffer solution (122) to the test strip in a distal position relative to the flow zone of terminal sample, causing delivery of the buffer solution conjugate that a second analyte binding agent is diffused proximally beyond the terminal sample flow zone to one or more test areas after the broadcast front distal sample diffuses proximal to one or more areas of assay by agglutinating the second analyte binding agent at analyte immobilized in the test zone, and detect the second binding agent of analytes immobilized in the test zone. 22. Method according to claim 21, in which the conjugate buffer solution is added to the test strip at same time as the sample is added to said test strip. 23. Method according to claim 21, in which the conjugate buffer solution is added to the test strip before the sample is added to said test strip. 24. Method according to claim 21, in which the conjugate buffer solution is added to the test strip after the sample is added to said test strip. 25. Procedure according to any of the claims 21 to 24, wherein the first binding agent of analytes do not bind to components in the sample other than the analyte 26. Procedure according to any of the claims 21 to 25, wherein the first binding agent of analytes are selected from the group consisting of antibodies, designer proteins, peptides, haptens, lysates containing the heterogeneous mixtures of antigens having binding sites of analytes, ligands and receptors. 27. Procedure according to any of the claims 21 to 26, wherein the second binding agent of analytes are contained in the test strip where the conjugate buffer solution, causing delivery of the solution conjugate buffer diffusion of the second binding agent of analytes 28. Procedure according to any of the claims 21 to 26, wherein the second binding agent of analytes is contained in the proximal test strip where delivers the conjugate buffer solution, delivering the conjugate buffer solution that diffusion of the second occurs analyte binding agent. 29. Procedure according to any of the claims 21 to 26, wherein the delivery of the buffer solution conjugated to the test strip includes delivery of the second analyte binding agent to the test strip within the conjugate buffer solution. 30. Procedure according to any of the claims 21 to 29, wherein the test zones comprise at least a first control zone with a binding agent of immobilized control, causing delivery of the buffer solution conjugated that a control agent diffuses proximally, more beyond the terminal sample flow zone, to the first zone of control and bind the control binder immobilized. 31. Procedure according to any of the claims 21 to 29, wherein the test zones comprise the first and second control zone, each comprising different amount of a control binder immobilized, delivering the conjugate buffer solution a control agent diffuses proximally, after the terminal sample flow zone, to the first and second zone of control and bind the control binder immobilized. 32. Procedure according to any of the claims 21 to 31, wherein the second binding agent of analytes is able to bind the components in the sample that do not Be the analyte. 33. Procedure according to any of the claims 21 to 31, wherein the second binding agent of analytes do not bind the components in the sample other than the analyte 34. Procedure according to any of the claims 21 to 33, wherein the second binding agent of analytes are labeled with a detectable marker, which detects the second analyte binding agent as well as the marker detectable 35. Procedure according to any of the claims 21 to 34, wherein the second binding agent of analytes is bound to a particle, detecting the second agent analyte binder as well as said particle.